Abstract
Many recombinant proteins carry an oligohistidine (His. X)-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion-nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.
Original language | English |
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Pages (from-to) | 31-38 |
Number of pages | 8 |
Journal | Methods |
Volume | 54 |
Issue number | 1 |
DOIs | |
State | Published - May 2011 |
Bibliographical note
Funding Information:This work was supported by NIH Grants GM27925 to J.E.H., NS-046242 and HL-566252 to S.W.W. and GM-070662 to M.G.F.
Keywords
- Analytical ultracentrifugation
- Fluorescence
- Histidine tag
- Nitrilotriacetic acid
- Tracer sedimentation
ASJC Scopus subject areas
- Molecular Biology
- General Biochemistry, Genetics and Molecular Biology